Long-term effects of peripubertal binge EtOH exposure on hippocampal microRNA expression in the rat

Abstract

Adolescent binge alcohol abuse induces long-term changes in gene expression, which impacts the physiological stress response and memory formation, two functions mediated in part by the ventral (VH) and dorsal (DH) hippocampus. microRNAs (miRs) are small RNAs that play an important role in gene regulation and are potential mediators of long-term changes in gene expression. Two genes important for regulating hippocampal functions include brain-derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT1), which we identified as putative gene targets of miR-10a-5p, miR-26a, miR-103, miR-495. The purpose of this study was to quantify miR-10a-5p, miR-26a, miR-103, miR-495 expression levels in the dorsal and ventral hippocampus of male Wistar rats during normal pubertal development and then assess the effects of repeated binge-EtOH exposure. In addition, we measured the effects of binge EtOH-exposure on hippocampal Drosha and Dicer mRNA levels, as well as the putative miR target genes, BDNF and SIRT1. Overall, mid/peri-pubertal binge EtOH exposure altered the normal expression patterns of all miRs tested in an age- and brain region-dependent manner and this effect persisted for up to 30 days post-EtOH exposure. Moreover, our data revealed that mid/peri-pubertal binge EtOH exposure significantly affected miR biosynthetic processing enzymes, Drosha and Dicer. Finally, EtOH-induced significant changes in the expression of a subset of miRs, which correlated with changes in the expression of their predicted target genes. Taken together, these data demonstrate that EtOH exposure during pubertal development has long-term effects on miRNA expression in the rat hippocampus.

Figure 1. Diagram of experimental paradigm.

Diagram depicting the age of sacrifice and specific treatment paradigms for each group of male Wistar rats. N = 10/group.

Figure 2. Peri-pubertal binge EtOH exposure alters miR expression during pubertal development in the dorsal hippocampus.

miR-10a-5p (A), miR-26a (B), miR-103 (C), and miR-495 (D) expression levels in untreated (solid line) and EtOH-treated (dashed line) pubertal male rats. N = 10/group. Data represent mean fold change ±SEM as compared to untreated PND 30 animals. Dissimilar letters indicate a statistically significant difference between groups (p<0.05).

Figure 3. Peri-pubertal binge EtOH alters miR expression during pubertal development in the ventral hippocampus.

miR-10a-5p (A), miR-26a (B), miR-103 (C), and miR-495 (D) expression levels in untreated (solid line) and EtOH-treated (dashed line) pubertal male rats. N = 10/group. Data represent mean fold change ±SEM as compared to untreated PND 30 animals. Dissimilar letters indicate a statistically significant difference between groups (p<0.05).

Figure 4. Peri-pubertal binge EtOH exposure differentially alters miR biosynthetic processing enzymes in the dorsal and ventral hippocampus.

Drosha (A, C) and Dicer (B, D) mRNA levels in the dorsal and ventral hippocampus in untreated (solid line) and EtOH-treated (dashed line) pubertal male rats. N = 10/group. Data represent mean fold change ±SEM as compared to untreated PND 30 animals. Dissimilar letters indicate a statistically significant difference between groups (p<0.05).

Figure 5. Diagram depicting predicted miR binding sites for BDNF and SIRT1.

Schematic diagram of the 3′UTR of (A) BDNF - 2,842 bp and (B) SIRT1- 1,607 bp. The putative binding sites for each miR were predicted using Targetscan (www.targetscan.org) and miRDB (www.miRDB.org) computer algorithm programs. The binding sites were predicted based on the presence of an 8-mer or 7-mer conserved miR seed sequence. Precise seed sequence positions are shown in parentheses.

Figure 6. Peri-pubertal binge EtOH exposure differentially alters miR target genes, BDNF and SIRT1, in the dorsal and ventral hippocampus.

BDNF (A, C) and SIRT1 (B, D) mRNA levels in the dorsal and ventral hippocampus in untreated (solid line) and EtOH-treated (dashed line) pubertal male rats. N = 10/group. Data represent mean fold change ±SEM as compared to untreated PND 30 animals. Dissimilar letters indicate a statistically significant difference between groups (p<0.05).

Figure 7. Peri-pubertal binge EtOH exposure did not affect circulating testosterone levels.

Plasma concentrations of testosterone (T) 60 min. after the last treatment. Data expressed as mean ±SEM T pg/ml. No statistically significant differences were observed.